Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

doi:10.1073/pnas.0401372101

Proapoptotic N-truncated BCL-xL protein activates endogenous mitochondrial channels in living synaptic terminals

Departments of ^‡‡Pharmacology and ^*Internal Medicine, Yale University School of Medicine, 333 Cedar Street, New Haven, CT 06520; ^‡Institut de Recherches Servier, 125 Chemin de Ronde, 78290 Croissy sur Seine, France; ^§Department of Molecular Microbiology and Immunology, Johns Hopkins University School of Public Health, 615 North Wolfe Street, Baltimore, MD 21205; ^¶Department of Pharmacology and Molecular Sciences, Johns Hopkins University School of Medicine, 725 North Wolfe Street, Baltimore, MD 21205; ^∥Laboratory of Cellular and Molecular Biophysics, National Institute of Child Health and Development, National Institutes of Health, 10 Center Drive, Bethesda, MD 20892; ^**Department of Basic Sciences, New York University College of Dentistry, New York, NY 10010; and ^†Marine Biological Laboratory, Woods Hole, MA 02543

Edited by Richard A. Flavell, Yale University School of Medicine, New Haven, CT, and approved July 29, 2004 (received for review February 26, 2004)

Abstract

Neuronal death is often preceded by functional alterations at nerve terminals. Anti- and proapoptotic BCL-2 family proteins not only regulate the neuronal death pathway but also affect excitability of healthy neurons. We found that exposure of squid stellate ganglia to hypoxia, a death stimulus for neurons, causes a cysteine protease-dependent loss of full-length antiapoptotic BCL-xL, similar to previous findings in mammalian cells. Therefore, to determine the direct effect of the naturally occurring proapoptotic cleavage product of BCL-xL on mitochondria, recombinant N-truncated BCL-xL was applied to mitochondria inside the squid presynaptic terminal and to purified mitochondria isolated from yeast. N-truncated BCL-xL rapidly induced large multi-conductance channels with a maximal conductance significantly larger than those produced by full-length BCL-xL. This activity required the hydrophobic C terminus and the BH3 domain of BCL-xL. Moreover, N-truncated BCL-xL failed to produce any channel activity when applied to plasma membranes, suggesting that a component of the mitochondrial membrane is necessary for its actions. Consistent with this idea, the large channels induced by N-truncated BCL-xL are inhibited by NADH and require the presence of VDAC, a voltage-dependent anion channel present in the outer mitochondrial membrane. These observations suggest that the mitochondrial channels specific to full-length and N-truncated BCL-xL contribute to their opposite effects on synaptic transmission, and are consistent with their opposite effects on the cell death pathway.

Footnotes

↵ †† To whom correspondence may be addressed. E-mail: hardwick{at}jhu.edu or leonard.kaczmarek{at}yale.edu.
This paper was submitted directly (Track II) to the PNAS office.
Abbreviations: VDAC, voltage-dependent anion channel; zVAD, benzyloxycarbonyl-Val-Ala-Asp; ANTS, 8-aminonapthalene-1,3,6–trisulfonic acid.